isoTarget: a genetic method for analyzing the functional diversity of splicing isoforms in vivo

Abstract

Protein isoforms generated by alternative splicing contribute to proteome diversity. Due to the lack of effective techniques, isoform-specific functions, expression, localization, and signaling mechanisms of endogenous proteins in vivo are unknown for most genes. Here we report a genetic method, termed isoTarget, for blocking the expression of a targeted isoform without affecting the other isoforms and for conditional tagging the targeted isoform for multi-level analyses in select cells. Applying isoTarget to two mutually exclusive isoforms of Drosophila Dscam, Dscam[TM1] and [TM2], we found that endogenous Dscam[TM1] is localized in dendrites while Dscam[TM2] is in both dendrites and axons. We demonstrate that the difference in subcellular localization between Dscam[TM1] and [TM2], rather than any difference in biochemical properties, leads to the two isoforms differential contributions to dendrite and axon development. Moreover, with isoTarget, we discovered that the subcellular enrichment of functional partners results in a DLK/Wallenda-Dscam[TM2]-Dock signaling cascade specifically in axons. isoTarget is an effective technique for studying how alternative splicing enhances proteome complexity.